Surface plasmon fluorescence microscopy characteristics and application to bioimaging

SURFACE PLASMON FLUORESCENCE MICROSCOPY: CHARACTERISTICS AND APPLICATION TO BIOIMAGING TANG WAI TENG (B Eng (Hons), NUS) A THESIS SUBMITTED FOR THE DEREE OF DOCTOR OF PHILOSOPHY IN COMPUTATION AND SYSTEMS BIOLOGY (CSB) SINGAPORE-MIT ALLIANCE NATIONAL UNIVERSITY OF SINGAPORE 2010 Acknowledgments First and foremost, thanks to my supervisors Prof Colin Sheppard and Prof Peter So for their guidance in my project, their patience and timely advice without which this work would not have been possible I am also grateful to Peter for the time I spent at So Lab during my exchange at MIT And not forgetting many thanks to the members of So Lab – Hyuk Sang Kwon, Euiheon Chung, Maxine Jonas, Jaewon Cha, Daekeun Kim, Heejin Choi, and Yanghyo Kim for making my stay a pleasant and memorable one Special thanks go to Euiheon who taught me about his standing wave TIRF system, and who has remained a wonderful collaborator and great friend Chapter six is a result of our collaboration together with Yanghyo I was fortunate to work with many wonderful people at Optical Bioimaging Lab – Kou Shan Shan, Naveen Balla, Shakil Rehman, Shalin Mehta, Elijah Yew, Zheng Wei, Si Ke, and Gong Wei Thanks to them for making the lab a fun and pleasant place to work in Our weekly Journal club was very enjoyable although our discussions always seemed to digress somehow Also, special thanks to Naveen and Sounderya Nagarajan for their help with cell culturing It was not always easy to find someone to coat the glass substrates I am thus indebted to Naganivetha Thiyagarajah and Xu Yingshun for their help with thin film deposition Finally, I am grateful to SMA for their support and funding over the past years for making this project possible Last but not least, I wish to thank my parents for their support while I complete my studies Table of Contents Table of Contents i Summary iv List of Tables vi List of Figures vii List of Symbols and Abbreviations x CHAPTER 1: INTRODUCTION .1 1.1 Motivation .1 1.2 Literature Review 1.3 Background 1.3.1 Total Internal Reflection Fluorescence (TIRF) .6 1.3.2 Surface Plasmon Resonance (SPR) .12 1.4 An overview 17 CHAPTER 2: MODELING SURFACE PLASMON-COUPLED EMISSION MICROSCOPY .18 2.1 Surface Plasmon-Coupled Emission 18 2.2 Description of Angular Spectrum Representation 22 2.3 A Model for SPCEM 23 2.3.1 Excitation of dipole in the object space 23 2.3.2 Electric field in the image space .25 2.3.3 Addition of a linear polarizer in the detection path .32 2.3.4 Intensity in image space 33 CHAPTER 3: EXPERIMENTAL RESULTS OF SPCEM .35 3.1 Simulation Parameters 36 i 3.2 Radiation Characteristics of SPCEM .38 3.2.1 P-polarized emission and fluorescence quenching 38 3.2.2 Back-focal plane image of SPCEM 41 3.3 Point Spread Function Characteristics of SPCEM 42 3.3.1 Electric field distributions .42 3.3.2 Calculated intensity point spread function 45 3.3.3 Experimental set-up of SPCEM 45 3.4 Discussion 51 CHAPTER 4: OPTICAL TRANSFER FUNCTION OF SURFACE PLASMONCOUPLED EMISSION MICROSCOPY 53 4.1 Vectorial Optical Transfer Function 53 4.2 Comparison between TIRFM and SPCEM .57 4.3 Deconvolution for SPCEM 60 CHAPTER 5: OPTICAL COMPENSATION FOR SURFACE PLASMONCOUPLED EMISSION MICROSCOPY 71 5.1 Theoretical Basis 72 5.2 Modification to SPCEM .75 5.2.1 Spiral phase plate 76 5.2.2 Polarization mode converter 78 5.2.3 Comparison of SPP and PMC 80 5.3 Experimental Results 87 5.3.1 Modified SPCEM imaging 92 CHAPTER 6: STANDING-WAVE SURFACE PLASMON-COUPLED EMISSION MICROSCOPY 98 6.1 Background 98 6.2 Theory 99 6.2.1 Creating standing wave with p-polarized light .103 6.2.2 SW-SPRF algorithm and resolution 108 6.3 Experimental Results 110 6.3.1 SW-SPRF set-up 110 ii CHAPTER 7: CONFOCAL SURFACE PLASMON RESONANCE FLUORESCENCE MICROSCOPY 117 7.1 SPR Excitation by Focused Beam 118 7.2 Theory for Confocal SPRF 125 7.3 Experimental Results 127 7.3.1 Generation of radially polarized beam 127 7.3.2 Confocal SPRF imaging 129 7.4 Detection with Conversion Element 132 CHAPTER 8: PERSPECTIVES AND CONCLUSION 139 BIBLIOGRAPHY 147 PUBLICATIONS 161 iii Summary The characteristics of imaging through a metal-coated glass cover slip using the TIRF configuration are examined This configuration makes use of surface plasmons to excite fluorescent molecules as well as to collect the emission light Also known as surface plasmon coupled emission microscopy (SPCEM), the advantages of this method include better background rejection and reduced photobleaching due to decreased fluorescence lifetime Through back and front focal plane imaging of subdiffraction-limit fluorescent beads, the experimental characteristics of surface plasmon-coupled emission microscopy are elucidated Furthermore, at the angle at which surface plasmon resonance occurs, it is shown that the emission of fluorescent beads collected through the metal layer results in an irregular point spread function that has a donut-like morphology with multiple rings extending out Based on the vectorial Debye-Wolf method, a model for the microscope is derived and used to compare with the experimental results In addition, the vectorial OTF of the microscope is obtained and it is shown to have zero crossings and negative values which contribute to the donut shape morphology Because of the distorted point spread function, point spread function engineering approaches or numerical deconvolution are necessary to compensate for the irregularity for practical use in cellular imaging Numerical deconvolution methods such as the Richardson-Lucy algorithm can compensate for the distortion However, an optical method is preferable because of less sensitivity to noise Due to iv the anisotropic and highly p-polarized emission characteristics, we proposed to engineer the point spread function using a conversion element, either a radial to linear mode converter or a spiral phase plate, which can compensate for the distortion Experimental results of compensated and uncompensated imaging using the conversion elements are presented It is shown that the conversion elements can restore the point spread function to one which is single-lobed, either by rotating the polarization of the emission light or by modifying its wavefront This change is beneficial for imaging Further improvements to the resolution of the microscope can be achieved by using standing surface plasmon resonance waves A sub-diffraction limited resolution of 120 nm is demonstrated by illuminating the sample with a standing wave and synthesizing the final image from three images of different phases Finally, a confocal-based surface plasmon microscope is demonstrated which employs radially polarized illumination This results in a tightly focused spot and better excitation Together with a spiral phase plate in the detection path, its detection efficiency is shown to be improved without sacrificing the lateral resolution of the microscope v List of Tables Table 3-1 Values used for numerical simulation 38 vi List of Figures Figure 1-1 Total internal reflection occurring at interface Figure 1-2 Total internal reflection with s- and p-polarized beams Figure 1-3 Intensity at interface while varying the angle of incidence 10 Figure 1-4 Plot of the penetration depth of the evanescent field 11 Figure 1-5 A metal and dielectric half-space supporting surface plasmons 13 Figure 1-6 Dispersion relation of surface plasmons 14 Figure 1-7 Geometry of the Kretschmann-Raether configuration 15 Figure 1-8 Right shift in the SPR reflectivity curve 16 Figure 2-1 Comparison of intensity enhancement 19 Figure 2-2 An objective-launched set-up for SPCE imaging 21 Figure 2-3 Excitation of dipole by p-polarized incident plane wave 24 Figure 2-4 A schematic view of the SPCEM imaging process 25 Figure 2-5 Axis convention used in the derivation of the field in medium 26 Figure 3-1 Contour plot of reflectivity 36 Figure 3-2 Family of reflectivity curves 37 Figure 3-3 Polar plot of the emission intensity of SPCEM 39 Figure 3-4 Plot of fluorescence collected by objective lens 40 Figure 3-5 Comparison of back-focal plane emission pattern of SPCEM 41 Figure 3-6 Electric field components for a perpendicular dipole 43 Figure 3-7 Electric field components for a parallel dipole 44 Figure 3-8 Intensity point spread function of SPCEM 45 vii Biss, D P., K S Youngworth, and T G Brown (2006), “Dark-field imaging with cylindrical-vector beams,” Appl Opt., 45(3), 470-479 Borejdo, J., N Calander, Z Gryczynski, and I Gryczynski (2006), “Fluorescence correlation spectroscopy in surface plasmon coupled emission microscope,” Opt Express, 14(17), 7878-7888 Borejdo, J., Z Gryczynski, N Calander, P Muthu, and I Gryczynski (2006), “Application of surface plasmon coupled emission to study of muscle,” Biophysical Journal, 91(7), 2626-2635 Born, M., and E Wolf (1999), Principles of optics: electromagnetic theory of propagation, interference and diffraction of light, 7th ed., Cambridge University Press, Cambridge; New York Bouhelier, A., F Ignatovich, A Bruyant, C Huang, G Colas des Francs, J Weeber, A Dereux, G P Wiederrecht, and L Novotny (2007), “Surface plasmon interference excited by tightly focused laser beams,” Opt Lett., 32(17), 25352537 Boutet de Monvel, J., S Le Calvez, and M Ulfendahl (2001), “Image restoration for confocal microscopy: improving the limits of deconvolution, with application to the visualization of the mammalian hearing organ,” Biophysical Journal, 80(5), 2455-2470 Bozhevolnyi, S., J Erland, K Leosson, P M Skovgaard, and J M Hvam (2001), “Waveguiding in surface plasmon polariton band gap structures,” Phys Rev Lett., 86(14), 3008-3011 Brian, A A., and H M McConnell (1984), “Allogeneic stimulation of cytotoxic T cells by supported planar membranes,” Proceedings of the National Academy of Sciences, 81, 6159-6163 Burghardt, T., J Charlesworth, M Halstead, J Tarara, and K Ajtai (2006), “In situ fluorescent protein imaging with metal film-enhanced total internal reflection microscopy,” Biophysical Journal, 90(12), 4662-4671 148 Campi, G (2005), “Actin and agonist MHC-peptide complex-dependent T cell receptor microclusters as scaffolds for signaling,” Journal of Experimental Medicine, 202(8), 1031-1036 Chance, R R., A Prock, and R Silbey (1974), “Lifetime of an excited molecule near a metal mirror: Energy transfer in the Eu3+⁄silver system,” J Chem Phys., 60(5), 2184-2185 Chon, J W M., X Gan, and M Gu (2002), “Splitting of the focal spot of a high numerical-aperture objective in free space,” Appl Phys Lett., 81(9), 15761578 Chon, J W M., and M Gu (2004), “Scanning total internal reflection fluorescence microscopy under one-photon and two-photon excitation: image formation,” Appl Opt., 43(5), 1063-1071 Chung, E., D Kim, Y Cui, Y Kim, and P T C So (2007), “Two-dimensional standing wave total internal reflection fluorescence microscopy: superresolution imaging of single molecular and biological specimens,” Biophysical Journal, 93(5), 1747-1757 Davis, J A., G H Evans, and I Moreno (2005), “Polarization-multiplexed diffractive optical elements with liquid-crystal displays,” Appl Opt., 44(19), 4049-4052 Dorn, R., S Quabis, and G Leuchs (2003), “Sharper focus for a radially polarized light beam,” Phys Rev Lett., 91(23), 2339011-2339014 Drexhage, K H (1970), “Influence of a dielectric interface on fluorescence decay time,” Journal of Luminescence, 1-2, 693-701 Dustin, M L et al (1998), “A novel adaptor protein orchestrates receptor patterning and cytoskeletal polarity in T-cell contacts,” Cell, 94(5), 667-677 Dustin, M L (2008), “T-cell activation through immunological synapses and kinapses,” Immunol Rev, 221, 77-89 149 Ebbesen, T W., H J Lezec, H F Ghaemi, T Thio, and P A Wolff (1998), “Extraordinary optical transmission through sub-wavelength hole arrays,” Nature, 391(6668), 667-669 Edel, J., M Wu, B Baird, and H G Craighead (2005), “High spatial resolution observation of single-molecule dynamics in living cell membranes,” Biophysical Journal, 88(6), L43-L45 Enderlein, J., and M Bohmer (2003), “Influence of interface dipole interactions on the efficiency of fluorescence light collection near surfaces,” Opt Lett., 28(11), 941-943 Evans, S D., H Allinson, N Boden, T M Flynn, and J R Henderson (1997), “Surface plasmon resonance imaging of liquid crystal anchoring on patterned self-assembled monolayers,” J Phys Chem B, 101(12), 2143-2148 Fägerstam, L G., Å Frostell-Karlsson, R Karlsson, B Persson, and I Rönnberg (1992), “Biospecific interaction analysis using surface plasmon resonance detection applied to kinetic, binding site and concentration analysis,” Journal of Chromatography A, 597(1-2), 397-410 Fang, N., H Lee, C Sun, and X Zhang (2005), “Sub-diffraction-limited optical imaging with a silver superlens,” Science, 308(5721), 534-537 Fiolka, R., M Beck, and A Stemmer (2008), “Structured illumination in total internal reflection fluorescence microscopy using a spatial light modulator,” Opt Lett, 33(14), 1629-1631 Ford, G W., and W Weber (1984), “Electromagnetic interactions of molecules with metal surfaces,” Physics Reports, 113(4), 195-287 Fuerhapter, S., A Jesacher, S Bernet, and M Ritsch-Marte (2005), “Spiral phase contrast imaging in microscopy,” Opt Express, 13(3), 689-694 Giebel, K., C Bechinger, S Herminghaus, M Riedel, P Leiderer, U Weiland, and M Bastmeyer (1999), “Imaging of cell/substrate contacts of living cells with surface plasmon resonance microscopy,” Biophysical Journal, 76(1), 509-516 150 Gonzalez, R (2002), Digital image processing, 2nd ed., Prentice Hall, Upper Saddle River N.J Grakoui, A., S K Bromley, C Sumen, M M Davis, A S Shaw, P M Allen, and M L Dustin (1999), “The immunological synapse: a molecular machine controlling T cell activation,” Science, 285(5425), 221-227 Gryczynski, I., J Malicka, Z Gryczynski, and J R Lakowicz (2004a), “Radiative decay engineering Experimental studies of surface plasmon-coupled directional emission,” Analytical Biochemistry, 324(2), 170-182 Gryczynski, I., J Malicka, Z Gryczynski, and J R Lakowicz (2004b), “Surface plasmon-coupled emission with gold films,” J Phys Chem B, 108(33), 12568-12574 Gryczynski, Z., J Borejdo, N Calander, E Matveeva, and I Gryczynski (2006), “Minimization of detection volume by surface-plasmon-coupled emission,” Analytical Biochemistry, 356(1), 125-131 Gu, M (1996), Principles of three dimensional imaging in confocal microscopes, World Scientific, Singapore; River Edge N.J Gu, M (2000), Advanced optical imaging theory, Springer, Berlin; New York He, R., G Chang, H Wu, C Lin, K Chiu, Y Su, and S Chen (2006), “Enhanced live cell membrane imaging using surface plasmon-enhanced total internal reflection fluorescence microscopy,” Opt Express, 14(20), 9307-9316 Hecht, E (2002), Optics, 4th ed., Addison-Wesley, Reading Mass Hellen, E H., and D Axelrod (1987), “Fluorescence emission at dielectric and metalfilm interfaces,” J Opt Soc Am B, 4(3), 337-350 151 Huang, X., P K Jain, I H El-Sayed, and M A El-Sayed (2007), “Plasmonic photothermal therapy (PPTT) using gold nanoparticles,” Lasers Med Sci., 23(3), 217-228 Hung, Y., I I Smolyaninov, C C Davis, and H Wu (2006), “Fluorescence enhancement by surface gratings,” Opt Express, 14(22), 10825-10830 Huppa, J B., and M M Davis (2003), “T-cell-antigen recognition and the immunological synapse,” Nat Rev Immunol, 3(12), 973-983 Irvine, D J., M A Purbhoo, M Krogsgaard, and M M Davis (2002), “Direct observation of ligand recognition by T cells,” Nature, 419(6909), 845-849 Kah, J C Y., T H Chow, B K Ng, S G Razul, M Olivo, and C J R Sheppard (2009), “Concentration dependence of gold nanoshells on the enhancement of optical coherence tomography images: a quantitative study,” Appl Opt., 48(10), D96-D108 Kano, H (2000), “A scanning microscope employing localized surface-plasmonpolaritons as a sensing probe,” Opt Commun., 182(1-3), 11-15 Kawata, S (2001), Near-field optics and surface plasmon polaritons, Springer Berlin Heidelberg, Berlin, Heidelberg Kitson, S C., W L Barnes, J R Sambles, and N P K Cotter (1996), “Excitation of molecular fluorescence via surface plasmon polaritons,” J Mod Opt., 43(3), 573-582 Krogsgaard, M., Q Li, C Sumen, J B Huppa, M Huse, and M M Davis (2005), “Agonist/endogenous peptide-MHC heterodimers drive T cell activation and sensitivity,” Nature, 434(7030), 238-243 Lakowicz, J R (2004), “Radiative decay engineering Surface plasmon-coupled directional emission,” Analytical Biochemistry, 324(2), 153-169 Lakowicz, J R (2005), “Radiative decay engineering 5: metal-enhanced fluorescence and plasmon emission,” Analytical Biochemistry, 337(2), 171-194 152 Lakowicz, J R., J Malicka, I Gryczynski, and Z Gryczynski (2003), “Directional surface plasmon-coupled emission: a new method for high sensitivity detection,” Biochemical and Biophysical Research Communications, 307(3), 435-439 Larkin, K G., D J Bone, and M A Oldfield (2001), “Natural demodulation of twodimensional fringe patterns I General background of the spiral phase quadrature transform,” J Opt Soc Am A, 18(8), 1862-1870 Lee, K et al (2003), “The immunological synapse balances T cell receptor signaling and degradation,” Science, 302(5648), 1218-1222 Lee, K., A D Holdorf, M L Dustin, A C Chan, P M Allen, and A S Shaw (2002), “T cell receptor signaling precedes immunological synapse formation,” Science, 295(5559), 1539-1542 Levene, M J., J Korlach, S W Turner, M Foquet, H G Craighead, and W W Webb (2003), “Zero-mode waveguides for single-molecule analysis at high concentrations,” Science, 299(5607), 682-686 Liedberg, B (1983), “Surface plasmon resonance for gas detection and biosensing,” Sensors and Actuators, 4(1), 299-304 Lin, A W H., N A Lewinski, J L West, N J Halas, and R A Drezek (2005), “Optically tunable nanoparticle contrast agents for early cancer detection: model-based analysis of gold nanoshells,” J Biomed Opt., 10(6), 064035 Liu, Z., H Lee, Y Xiong, C Sun, and X Zhang (2007), “Far-field optical hyperlens magnifying sub-diffraction-limited objects,” Science, 315(5819), 1686-1686 Liu, Z., Q Wei, and X Zhang (2005), “Surface plasmon interference nanolithography,” Nano Lett., 5(5), 957-961 Loo, C., A Lowery, N Halas, J West, and R Drezek (2005), “Immunotargeted nanoshells for integrated cancer imaging and therapy,” Nano Lett., 5(4), 709711 153 Lucy, L B (1974), “An iterative technique for the rectification of observed distributions,” Astronomical Journal, 79, 745-747 Lukosz, W (1966), “Optical Systems with Resolving Powers Exceeding the Classical Limit,” J Opt Soc Am., 56(11), 1463-1471 Lukosz, W., and R E Kuhn (1977), “Fluorescence lifetime of magnetic and electric dipoles near a dielectric interface,” Optics Communications, 20(2), 195-199 Mair, A., A Vaziri, G Weihs, and A Zeilinger (2001), “Entanglement of the orbital angular momentum states of photons,” Nature, 412(6844), 313-316 Malicka, J., I Gryczynski, Z Gryczynski, and J R Lakowicz (2003), “DNA hybridization using surface plasmon-coupled emission,” Analytical Chemistry, 75(23), 6629-6633 Matveeva, E., Z Gryczynski, I Gryczynski, and J R Lakowicz (2004), “Immunoassays based on directional surface plasmon-coupled emission,” Journal of Immunological Methods, 286(1-2), 133-140 Matveeva, E., Z Gryczynski, I Gryczynski, J Malicka, and J R Lakowicz (2004), “Myoglobin immunoassay utilizing directional surface plasmon-coupled emission,” Analytical Chemistry, 76(21), 6287-6292 McConnell, H M., T H Watts, R M Weis, and A A Brian (1986), “Supported planar membranes in studies of cell-cell recognition in the immune system,” Biochim Biophys Acta, 864(1), 95-106 Moh, K J., X Yuan, J Bu, S W Zhu, and B Z Gao (2008), “Surface plasmon resonance imaging of cell-substrate contacts with radially polarized beams,” Opt Express, 16(25), 20734-20741 Moh, K J., X Yuan, J Bu, S W Zhu, and B Z Gao (2009), “Radial polarization induced surface plasmon virtual probe for two-photon fluorescence microscopy,” Opt Lett., 34(7), 971-973 154 Monks, C R., B A Freiberg, H Kupfer, N Sciaky, and A Kupfer (1998), “Threedimensional segregation of supramolecular activation clusters in T cells,” Nature, 395(6697), 82-86 Nanophoton Corp (2010), Nanophoton Corporation, Z polarization device (ZPol) Neil, M A A., R Juskaitis, and T Wilson (1997), “Method of obtaining optical sectioning by using structured light in a conventional microscope,” Opt Lett., 22(24), 1905-1907 O'Keefe, J P., and T F Gajewski (2005), “Cutting edge: cytotoxic granule polarization and cytolysis can occur without central supramolecular activation cluster formation in CD8+ effector T cells,” J Immunol, 175(9), 5581-5585 Palik, E D (1997), Handbook of optical constants of solids, Academic Press Pawley, J (2006), Handbook of biological confocal microscopy, 3rd ed., Springer, New York N.Y Pendry, J (2000), “Negative refraction makes a perfect lens,” Phys Rev Lett., 85(18), 3966-3969 Piliarik, M., H Vaisocherová, and J Homola (2005), “A new surface plasmon resonance sensor for high-throughput screening applications,” Biosensors and Bioelectronics, 20(10), 2104-2110 Purbhoo, M A., D J Irvine, J B Huppa, and M M Davis (2004), “T cell killing does not require the formation of a stable mature immunological synapse,” Nat Immunol, 5(5), 524-530 Raether, H (1988), Surface plasmons on smooth and rough surfaces and on gratings, Springer-Verlag, Berlin; New York Richards, B., and E Wolf (1959), “Electromagnetic diffraction in optical systems II Structure of the image field in an aplanatic system,” Proc Roy Soc London A, 253(1274), 358-379 155 Richardson, W (1972), “Bayesian-based iterative method of image restoration,” J Opt Soc Am., 62(1), 55-59 Rothenhäusler, B., and W Knoll (1988), “Surface-plasmon microscopy,” Nature, 332(6165), 615-617 Saito, T., and T Yokosuka (2006), “Immunological synapse and microclusters: the site for recognition and activation of T cells,” Curr Opin Immunol, 18(3), 305-313 Schmelzeisen, M., J Austermann, and M Kreiter (2008), “Plasmon mediated confocal dark-field microscopy,” Opt Express, 16(22), 17826-17841 Schmid, E L., A P Tairi, R Hovius, and H Vogel (1998), “Screening ligands for membrane protein receptors by total internal reflection fluorescence: the 5HT3 serotonin receptor,” Anal Chem., 70(7), 1331-1338 Schönle, A., and S W Hell (2002), “Calculation of vectorial three-dimensional transfer functions in large-angle focusing systems,” J Opt Soc Am A, 19(10), 2121-2126 Schurig, D., J J Mock, B J Justice, S A Cummer, J B Pendry, A F Starr, and D R Smith (2006), “Metamaterial electromagnetic cloak at microwave frequencies,” Science, 314(5801), 977-980 Selvaraj, P., M L Plunkett, M Dustin, M E Sanders, S Shaw, and T A Springer (1987), “The T lymphocyte glycoprotein CD2 binds the cell surface ligand LFA-3,” Nature, 326(6111), 400-403 Sentenac, A., P Chaumet, and K Belkebir (2006), “Beyond the Rayleigh criterion: grating assisted far-field optical diffraction tomography,” Phys Rev Lett., 97(24), 2439011-2439014 Sheppard, C J R., and A Choudhury (2004), “Annular Pupils, Radial Polarization, and Superresolution,” Appl Opt., 43(22), 4322 156 Sheppard, C J R., and K C Larkin (1997), “Vectorial pupil functions and vectorial transfer functions,” Optik, 107(2), 79-87 Simpson, N B., L Allen, and M J Padgett (1996), “Optical tweezers and optical spanners with Laguerre-Gaussian modes,” J Mod Opt., 43(12), 2485-2491 Slavík, R., and J Homola (2007), “Ultrahigh resolution long range surface plasmonbased sensor,” Sensors and Actuators B: Chemical, 123(1), 10-12 Smolyaninov, I I., Y Hung, and C C Davis (2007), “Magnifying superlens in the visible frequency range,” Science, 315(5819), 1699-1701 So, P T., H S Kwon, and C Y Dong (2001), “Resolution enhancement in standingwave total internal reflection microscopy: a point-spread-function engineering approach,” J Opt Soc Am A, 18(11), 2833-2845 Somekh, M G (2002), “Surface plasmon fluorescence microscopy: an analysis,” J Microsc., 206(2), 120-131 Sönnichsen, C., B M Reinhard, J Liphardt, and A P Alivisatos (2005), “A molecular ruler based on plasmon coupling of single gold and silver nanoparticles,” Nat Biotechnol., 23(6), 741-745 Stabler, G., M G Somekh, and C W See (2004), “High-resolution wide-field surface plasmon microscopy,” J Microsc., 214(3), 328-333 Stalder, M., and M Schadt (1996), “Linearly polarized light with axial symmetry generated by liquid-crystal polarization converters,” Opt Lett., 21(23), 19481950 Stefani, F., K Vasilev, N Bocchio, N Stoyanova, and M Kreiter (2005), “Surfaceplasmon-mediated single-molecule fluorescence through a thin metallic film,” Phys Rev Lett., 94(2), 0230051-0230054 Steyer, J A., H Allinson, and W Almers (1997), “Transport, docking and exocytosis of single secretory granules in live chromaffin cells,” Nature, 388(6641), 474478 157 Sund, S., and D Axelrod (2000), “Actin dynamics at the living cell submembrane imaged by total internal reflection fluorescence photobleaching,” Biophysical Journal, 79(3), 1655-1669 Takai, Y., M L Reed, S J Burakoff, and S H Herrmann (1987), “Direct evidence for a receptor-ligand interaction between the T-cell surface antigen CD2 and lymphocyte-function-associated antigen 3,” Proc Natl Acad Sci U.S.A, 84(19), 6864-6868 Tanaka, T., and S Kawata (2005), “Real-time observation of birefringence by laserscanning surface plasmon resonance microscope,” Opt Express, 13(18), 69056911 Tanaka, T., and S Yamamoto (2003), “Laser-scanning surface plasmon polariton resonance microscopy with multiple photodetectors,” Appl Opt., 42(19), 4002-4007 Tang, W T., E Chung, Y Kim, P T C So, and C J R Sheppard (2007), “Investigation of the point spread function of surface plasmon-coupled emission microscopy,” Opt Express, 15(8), 4634-4646 Tidwell, S C., D H Ford, and W D Kimura (1990), “Generating radially polarized beams interferometrically,” Appl Opt., 29(15), 2234-2239 Török, P., P D Higdon, and T Wilson (1998), “Theory for confocal and conventional microscopes imaging small dielectric scatterers,” J Mod Opt., 45(8), 1681-1698 Török, P., and C J R Sheppard (2002), “The role of pinhole size in high-aperture two- and three-photon microscopy,” in Confocal and two-photon microscopy: foundations, applications, and advances, pp 127-151, Wiley-Liss, New York Török, P., and P Varga (1997), “Electromagnetic diffraction of light focused through a stratified medium,” Appl Opt., 36(11), 2305-2312 158 Varma, R., G Campi, T Yokosuka, T Saito, and M L Dustin (2006), “T Cell Receptor-Proximal Signals Are Sustained in Peripheral Microclusters and Terminated in the Central Supramolecular Activation Cluster,” Immunity, 25(1), 117-127 Vasilev, K., F D Stefani, V Jacobsen, W Knoll, and M Kreiter (2004), “Reduced photobleaching of chromophores close to a metal surface,” J Chem Phys., 120(14), 6701-6704 Veselago, V G (1968), “The electrodynamics of substances with simultaneous negative values of epsilon and mu,” Soviet Physics Uspekhi, (4), 509-514 Watts, T H., A A Brian, J W Kapples, P Marrack, and H M McConnell (1984), “Antigen presentation by supported planar membranes containing affinitypurified I-Ad,” Proceedings of the National Academy of Sciences, 81(23), 7564-7568 Weeber, J., A Dereux, J R Krenn, and J Goudonnet (1999), “Plasmon polaritons of metallic nanowires for controlling submicron propagation of light,” Phys Rev B, 60(12), 9061-9068 Weyl, H (1919), “Ausbreitung elektromagnetischer Wellen uber einem ebenen Leiter,” Annalen der Physik, 365(21), 481-500 Wolf, E (1959), “Electromagnetic diffraction in optical systems I An integral representation of the image field,” Proc Roy Soc London A, 253(1274), 349357 Yamaguchi, R., T Nose, and S Sato (1989), “Liquid crystal polarizers with axially symmetrical properties,” Jpn J Appl Phys., 28(Part 1, No 9), 1730-1731 Ye, C (1995), “Construction of an optical rotator using quarter-wave plates and an optical retarder,” Opt Eng., 34(10), 3031-3035 Yeatman, E., and E A Ash (1987), “Surface plasmon microscopy,” Electron Lett., 23(20), 1091-1092 159 Yokosuka, T., and T Saito (2010), “The immunological synapse, TCR microclusters, and T cell activation,” Curr Top Microbiol Immunol, 340, 81-107 Yokosuka, T., K Sakata-Sogawa, W Kobayashi, M Hiroshima, A Hashimoto-Tane, M Tokunaga, M L Dustin, and T Saito (2005), “Newly generated T cell receptor microclusters initiate and sustain T cell activation by recruitment of Zap70 and SLP-76,” Nat Immunol, 6(12), 1253-1262 Yokota, H., K Saito, and T Yanagida (1998), “Single molecule imaging of fluorescently labeled proteins on metal by surface plasmons in aqueous solution,” Phys Rev Lett., 80(20), 4606-4609 Yoshiki, K., K Ryosuke, M Hashimoto, T Araki, and N Hashimoto (2007), “Second-harmonic-generation microscope using eight-segment polarizationmode converter to observe three-dimensional molecular orientation,” Opt Lett., 32(12), 1680-1682 160 Publications Journals W T Tang, E Chung, Y Kim, C J R Sheppard, P T C So, “Investigation of the point spread function of surface plasmon coupled emission microscopy”, Opt Express, Vol 15, No 8, 4634-4646 (2007) W T Tang, E Chung, Y Kim, C J R Sheppard, P T C So, “Effects of using a metal layer in total internal reflection fluorescence microscopy”, Appl Phys A, 89, 333-335 (2007) W T Tang, Elijah Y S Yew, C J R Sheppard, “Polarization conversion in confocal microscopy with radially polarized illumination, Opt Lett 34, 2147 (2009) C J R Sheppard, S Rehman, N K Balla, Elijah Y S Yew, W T Tang, “Bessel beams: effects of polarization,” Opt Comm 282, 4647-4656 (2009) E Chung, Y Kim, W T Tang, C J R Sheppard, P T C So, “Wide-field extendedresolution fluorescence microscopy with standing surface-plasmon-resonance waves,” Opt Lett 34(15), 2366 (2009) W T Tang, E Chung, Y Kim, C J R Sheppard, P T C So, “Surface plasmoncoupled emission microscopy with a spiral phase plate”, Opt Lett., 35, 517-519 (2010) Conferences W T Tang*, E Chung, Y Kim, P T C So, C J R Sheppard, “Effects of using a metal layer in total internal reflection fluorescence microscopy,” International Workshop on Plasmonics and Applications in Nanotechnologies, Singapore (2006) E Chung*, W T Tang, Y Kim, C J R Sheppard, P T C So, “Towards the standing wave surface plasmon resonance fluorescence microscopy”, Photonics West, Plasmonics in Biology and Medicine, San Jose, USA (2007) W T Tang*, E Chung, Y Kim, P T C So, C J R Sheppard, “Surface plasmon microscopy”, 5th International Symposium on Nanomanufacturing (ISNM-5), Singapore (2008) 161 W T Tang*, E Chung, Y Kim, P T C So, C J R Sheppard, “Surface plasmon enhancement of total internal reflection fluorescence for biosensors”, Optics within Life Sciences 10, Biophotonics Asia 2008, Singapore (2008) W T Tang*, P T C So, C J R Sheppard, “Surface plasmon coupled emission microscopy and its vectorial optical transfer function”, The 4th Asian and Pacific Rim Symposium on Biophotonics, Jeju, Korea (2009) W T Tang, C J R Sheppard*, “Application of spiral phase plate in surface plasmon microscopy”, Focus on Microscopy, Shanghai, China (2010) 162 ... many areas mentioned above, to date there have not been many studies devoted to understanding the application of surface plasmons to bioimaging, much less to fluorescence microscopy In many of the... investigators mainly made use of the reflectivity and transmission characteristics of surface plasmons But the use of surface plasmons involving fluorescence excitation and emission in microscopy. .. therefore to study the effects of using surface plasmons specifically to fluorescence imaging and to elucidate the properties of such an imaging modality in order to properly understand its characteristics